Interferons are now widely recognized as being an important group of biological proteins capable of rendering cells or whole organisms resistant to virus attack. Furthermore, interferons have been proven to play a significant role in the regulation of the immune system in vivo and in vitro and exhibit numerous other so-called non-viral activities such as antiprotozoal and antibacterial activities, antiproliferative activity of tumor cells, potentiation of the natural killer cell system, and potentiation of the Human Leukocyte Antigen (HLA) System. As such, it is believed interferons are proteins with tremendous potential in the clinical area.
The clinical and therapeutic application of interferon, however, has been slow due to both the high cost of obtaining human interferon and the inability to obtain pure human interferon. Additionally, recent developments in interferon research revealed that several different classes of human interferons exist. Specifically, three different classes of interferon have been demonstrated: leukocyte (HUIFN-.alpha.), fibroblast (HUIFN-.beta.) and immune (HUIFN-gamma) interferons. These classes correspond to the cell type employed as the source of human interferon, those being buffy coat leukocytes, fibroblasts and immunocompetent lymphocytes, respectively. Each of these classes in turn have been shown to demonstrate variable activities with respect to the aforementioned viral and non-viral activities attributed to interferon generally. It has, therefore, become desirable to develop the ability to isolate and generate pure interferon of each of the designated classes. Attempts to isolate each of these interferon classes, however, have been further complicated by the discovery that within, for example, the leukocyte interferon class, as many as thirteen species exist.
Whereas classical techniques for protein purification, such as gel filtration, high performance liquid chromatography and antibody affinity chromatography employing polyclonal antibodies specific to interferons, have been employed to isolate and partially characterize the aforementioned classes and intra-class species of human interferon, these techniques cannot be economically employed to isolate sufficient quantities of all pure interferon species for pharmaceutical and/or diagnostic use. There is, therefore, a critical need to develop a means for economically isolating the pure human interferon species.
Employment of the new hybridoma technology for monoclonal antibody production should provide a means for economically isolating large quantities of pure human interferons. The hybridoma technique for producing monoclonal antibodies, as originally described by Kohler and Milstein, comprises fusing spleen lymphocytes with malignant cells (myelomas) derived from a malignant cell line so as to create a fused cell hybrid cell line which possesses charcteristics of both the lymphocytes and myeloma cells (See Kohler and Milstein, Nature 256: 496-497 (1975)). The fused cell hybrids, called hybridomas, produce and secrete a single type of immunoglobulin (antibody) and are immortal. The combination of these two features allows for the continuous and reproducible production of a single tye of antibody molecule by the hybrid cells. The traditional techniques of immunizing an animal with an immunogen (usually consisting of the desired antigen together with impurities, each antigen having a variable number of epitopes) and thereafter collecting the sera from the immunized animal yield a mixture of many different antibodies having different specificities (the ability to recognize or bind with certain epitopes). Antibodies to antigenic impurities in the immunogenic preparation may also be developed by the immunized animal. Moreover, this mixture of antibodies can almost never be reproduced identically.
In both the traditional and hybridoma methods for producing antibodies, the specificity of the antibodies produced thereby depends upon the antigen or antigens presented (used to immunize) to the animal. Therefore, the traditional technique often produces a mixture of desired and undesired antibodies and it may be difficult to separate out the undesired portion. The hybridoma technique makes it possible to produce pure antibody preparation.
Recently, various monoclonal antibody preparations have been produced which have been claimed to be specific to various species of human interferon-alpha (HUIFN-.alpha.). As is discussed below, however, none of these antibody preparations contain antibodies capable of simultaneously recognizing all thirteen known species of human interferon-alpha.
U.S. Pat. No. 4,423,147 to Secher et al. and the publication by Secher and Burke, Nature 285: 446 (1980), discloses the preparation of a monoclonal antibody specific to one species of HUIFN-.alpha., that being a species having a molecular weight of about 18,000. The monoclonal antibody generated in the '147 patent was produced by first immunizing mice with a crude preparation of lymphoblastoid interferon and thereafter fusing antigen primed mouse spleen lymphocytes with myeloma cells to create a hybridoma cell line.
The publications by Staehelin et al., Proceedings of the National Academy of Sciences 78: 1848, Imai et al., Journal of Immunology 128: 2824-2825, and Novick et al., Journal of Immunology 129: 2244 (1982), disclose the preparation of hybridoma cell lines, each of which produce a monoclonal antibody to but a single species of recombinant interferons or only a few species, of native HUIFN-a.
In order to provide for the economical clinical application of HUIFN-.alpha. comprising all species of HUIFN-.alpha., it became, therefore, desirable to develop monoclonal antibodies which will recognize all native species of the HUIFN-.alpha. class and develop a process by which such antibodies could be produced. A monoclonal antibody has now, surprisingly, been made having the non-expected property of capability of binding all twelve native species of HUIFN-.alpha.. This increased recognition capability represents a significant improvement to the art.